Effective use of transgenes for the treatment of inherited and acquired disorders requires efficient delivery of transgenes. Various vector systems have been developed that are capable of delivering a transgene to a target cell. However, there remains a need to improve efficiency of available gene transfer methods. Improved efficiency is desirable both to increase the ability of the vector to correct the cellular defect, and to decrease the required amount of the vector and thereby reduce toxicity.
Adenoviral vectors, have been designed to take advantage of the desirable features of adenovirus which render it a suitable vehicle for nucleic acid transfer. Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb, which has been well-characterized through studies in classical genetics and molecular biology (Horwitz, M. S., "Adenoviridae and Their Replication," in Virology, 2nd edition, Fields et al., eds., Raven Press, New York, 1990). The viral genes are classified into early (known as E1-E4) and late (known as L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. The demarcation between these events is viral DNA replication. The human adenoviruses are divided into numerous serotypes (approximately 47, numbered accordingly and classified into 6 subgroups: A, B, C, D, E and F), based upon properties including hemaglutination of red blood cells, oncogenicity, DNA base and protein amino acid compositions and homologies, and antigenic relationships.
Recombinant adenoviral vectors have several advantages for use as gene transfer vectors, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapv 1:51-64, 1994).
The cloning capacity of an adenovirus vector is proportional to the size of the adenovirus genome present in the vector. For example, a cloning capacity of about 8 kb can be created from the deletion of certain regions of the virus genome dispensable for virus growth, e.g., E3, and the deletion of a genomic region such as E1 whose function may be restored in trans from 293 cells (Graham, F. L., J. Gen. Virol. 36:59-72, 1977) or A549 cells (Imler et al., Gene Therapy 3:75-84, 1996). Such E1 deleted vectors are rendered replication-defective. The upper limit of vector DNA capacity for optimal carrying capacity is about 105%-108% of the length of the wild-type genome. Further adenovirus genomic modifications are possible in vector design using cell lines which supply other viral gene products in trans, e.g., complementation of E2a (Zhou et al., J. Virol. 70:7030-7038, 1996), complementation of E4 (Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995; Wang et al., Gene Ther. 2:775-783, 1995), or complementation of protein IX (Caravokyri et al., J. Virol. 69:6627-6633, 1995; Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995).
Adenoviral vectors for use in gene transfer to cells and in gene therapy applications commonly are derived from adenoviruses by deletion of the early region 1 (E1) genes (Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992). Deletion of E1 genes renders the vector replication defective and significantly reduces expression of the remaining viral genes present within the vector. However, it is believed that the presence of the remaining viral genes in adenovirus vectors can be deleterious to the transfected cell for one or more of the following reasons: (1) stimulation of a cellular immune response directed against expressed viral proteins, (2) cytotoxicity of expressed viral proteins, and (3) replication of the vector genome leading to cell death.
Transgenes that have been expressed to date by adenoviral vectors include p53 (Wills et al., Human Gene Therapy 5:1079-188, 1994); dystrophin (Vincent et al., Nature Genetics 5:130-134, 1993; erythropoietin (Descamps et al., Human Gene Therapy 5:979-985, 1994; omithine transcarbamylase (Stratford-Perricaudet et al., Human Gene Therapy 1:241-256, 1990; We et al., J. Biol. Chem. 271:3639-3646, 1996;); adenosine deaminase (Mitani et al., Human Gene Therapy 5:941-948, 1994); interleukin-2 (Haddada et al., Human Gene Therapv 4:703-711, 1993); and 60 1-antitrypsin (Jaffe et al., Nature Genetics 1:372-378, 1992); thrombopoietin (Ohwada et al., Blood 88:778-784, 1996); and cytosine deaminase (Ohwada et al., Hum. Gene Ther. 7:1567-1576, 1996).
The particular tropism of adenoviruses for cells of the respiratory tract has relevance to the use of adenovirus in gene transfer for cystic fibrosis (CF), which is the most common autosomal recessive disease in Caucasians. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that disturb the cAMP-regulated Cl.sup.- channel in airway epithelia result in pulmonary dysfumction (Zabner et al., Nature Genetics 6:75-83, 1994). Adenovirus vectors engineered to carry the CFTR gene have been developed (Rich et al., Human Gene Therapy 4:461-476, 1993) and studies have shown the ability of these vectors to deliver CFTR to nasal epithelia of CF patients (Zabner et al., Cell 75:207-216, 1993), the airway epithelia of cotton rats and primates (Zabner et al., Nature Genetics 6:75-83, 1994), and the respiratory epithelium of CF patients (Crystal et al., Nature Genetics 8:42-51, 1994). Recent studies have shown that administering an adenoviral vector containing a DNA sequence encoding CFTR to airway epithelial cells of CF patients can restore a functioning chloride ion channel in the treated epithelial cells (Zabner et al., J. Clin. Invest. 97:1504-1511, 1996; U.S. Pat. No. 5,670,488, issued Sep. 23, 1997).
Transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia of patients with cystic fibrosis (CF) thus provides an example of successful use of gene transfer to correct a cellular defect, i.e., the CF defect in electrolyte transport. Vector systems including adenoviral vectors (Zabner et al. (1993) Cell 75: 207; Knowles et al. (1995) New Engl. J. Med. 333: 823; Hay et al. (1995) Hum. Gene. Ther. 6: 1487; Zabner et al. (1996) J. Clin. Invest. 97: 1504 and U.S. Pat. No. 5,670,488) and cationic lipids (Caplen et al. (1995) Nat. Med. 1: 39 and U.S. Pat. No. 5,650,096) have been shown to be capable of transferring the CFTR cDNA and expressing CFTR in mature ciliated human airway epithelia. The successful delivery of CFTR in such cells is manifest in the appearance of a functional chloride ion channel in the treated cells.
While CFTR cDNA can be delivered to target cells for expression, current adenoviral vectors are less than optimal in delivering the CFTR cDNA to airway epithelia because the binding of the virus to the apical (exposed) surface of the epithelium is limited. Grubb et al. (1994) Nature 371: 802. The limited infection can be partially overcome by increasing the contact time between the virus and the apical surface. Zabner et al. (1996) J. Virol. 70: 6994.
Cationic lipid vector-mediated gene transfer to mature human airway epithelia is also suboptimal. Caplen et al. (1995) Nat. Med. 1: 39. While it appears that cationic molecules bind to the cell surface and in some cases are taken up by the cell, important barriers to transgene expression may be release of DNA from the endosome, entry into the nucleus, release of DNA from the cationic molecule, and transcription of the DNA. Zabner et al. (1995) J. Biol. Chem. 270: 18997.
Gene transfer systems that combine viral and nonviral components have been reported. Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90: 11548; Wu et al. (1994) J. Biol. Chem. 269: 11542; Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6099; Yoshimura et al. (1993) J. Biol. Chem. 268: 2300; Curiel et al. (1991) Proc. Natl. Acad. Sci USA 88: 8850; Kupfer et al. (1994) Hum. Gene Ther. 5: 1437; and Gottschalk et al. (1994) Gene Ther. 1: 185. In most cases, adenovirus has been incorporated into the gene delivery systems to take advantage of its endosomolytic properties. The reported combinations of viral and nonviral components generally involve either covalent attachment of the adenovirus to a gene delivery complex or co-internalization of unbound adenovirus with cationic lipid: DNA complexes. Further, the transferred gene is contained in plasmid DNA that is exogenous to the adenovirus. In these formulations, large amounts of adenovirus are required, and the increases in gene transfer are often modest.
Calcium phosphate is often used to facilitate entry of DNA per se into cells in transformation or transfection procedures. Transfection with calcium phosphate co-precipitates containing plasmid DNA or naked adenoviral DNA is well known for cultured cell lines (Chen et al. (1987) Mol. Cell. Biol. 7:2745-2752; Graham et al. (1973) Virol. 52: 465-477). However, such DNA calcium phosphate co-precipitates are generally ineffective in primary cell cultures and in vivo. To date, there have been no reports of calcium phosphate being used to enhance transfection with recombinant viral vectors, including adenovirus.
Accordingly, there is a need in the art for improved vector systems for the efficient delivery of transgenes to target cells. The present invention overcomes certain limitations associated with adenoviral vectors and while retaining the desirable features of the vector system.